20.109(S10): TA notes for module 2

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General notes

Key preparation:

  • Lots of wild-type inverse pericam plasmid (in the pRSET vector) must be available.
  • Mutant IPC plasmid (M124S) should also be prepared in advance.
  • Need to streak out DE3 and DE3/IPC(wild-type) from frozen stocks in advance of liquid culture setup.
    • To be most careful, may want to freshly transform DE3 with pRSET-IPC miniprep.
    • DE3 in collection is NB301/AB2
    • DE3 w/IPC is NB303/AB4

Scheme: each pair of students will make two protein mutants, and test two candidate colonies per mutant. Specifically, students will choose only one mutation of their own, and run a 'positive control' mutation in parallel.

Daily Notes

Day 1

Materials required:

  1. None: all work today is computer work.

Day of Lab (T/W):

  • No quiz.
  • Primers for mutagenesis must be ordered right away, rush delivery!
  • Enzymes for diagnostics may need to be ordered as well, check designs.

How it went:

  • When this is not run as the first module (as it was in S09), there is a bit more time available for design, and the original CaM sequence exploration from S08 should be used, rather than the pre-highlighted Word document. This may help students realize that one bp of CaM has been deleted, for whatever reason, in the IPC sequence.
  • This year I had students check with me how far apart their new restriction site is from any existing same restriction sites in the plasmid; they are not really poised to do that calculation alone yet. I just have a simple Excel calculation set up for this purpose.

Day 2

Materials required:

  1. RT water for primer resuspension
  2. Quick-Change SDM kit. 1 reaction per student, plus 1 control reaction, plus spare reagents (ideally).
    • cat # 200519

Day of Lab (T/W):

  • Quiz (prepared by TA)
  • Prepare PCR tubes on racks obtained from the freezer
  • Get primers (forward and reverse) ready just before lab lecture ends
  • Prepare a few aliquots of Master Mix for students, plus a control reaction:
    • Each mutagenesis reaction should have 5 μL of buffer, 1 μL of dNTPs, and 37 μL of water, for a total of 43 μL.
    • See googledoc for total calculations
    • Control rxn. is: 43 μL Master Mix, 2 μL DNA, 1.25 μL each primer, and thus 2.5 μL extra water.
  • Guide journal article discussion (assign figures at beginning of class).
  • At end of day: freeze SDM DNA

How it went:

  • This year, the teaching sample initially gave no colonies, and we realized that the student samples weren't going to either. Although a 1:200 dilution of miniprep has always worked during the past two years, this year we had to titrate to 1:300--1:400 to get colonies. All student samples were re-done by us over the weekend. As usual, we parlayed a setback into a teachable moment, explaining how we determined what the most likely reason for the reaction failure was (e.g., we saw that the positive control that came with the kit worked just fine, suggesting something specific to our DNA vs. general reaction components).

Day 3

Materials required:

  1. Agarose gel electrophoresis
    • 1% agarose gels, up to 6 groups can fit per gel
    • TAE buffer
    • 1 Kb ladder
    • Post sample table at gel bench, put out nitrile gloves
  2. Bacterial transformation
    • LB+Amp plates - > 1.5 l of LB requires 45 min of autoclave and is hard to pour, should separate into 2 flasks for 30 min of autoclave
    • autoclaved glass tubes
    • LB broth
    • 1000X ampicillin - central stock per day
    • competent XL1-Blue cells (come with SDM kit)

Day of Lab (T/W)

  • Pre-warm water bath to 42 C, tube racks, etc.
  • Teaching faculty should prepare one positive control plate (in addition to the ones made by the students).

Days after Lab (W/R):

  • On W/R check student plates, pluck two colonies per mutant plate, and grow liquid O/N cultures. (Amp only, no Cam yet!)
  • Students with no colonies will be given their choice of any student candidate. (Some check on repeatability this way.)

How it went:

All but two groups got some colonies. A couple of groups only got a few colonies, but enough to proceed with.

Day 4

Materials required:

  1. Sub-culture DE3 in the morning.
    • Need 1x5mL tubes per pair, plus two extra to be safe.
    • Typical sub-culture: from OD > 2 to OD = 0.1 may take ~3-5 hours to reach OD = 0.6. Sub-culture to 0.15 or even 0.2 makes for shorter and more predictable lag phase - go with that! Stagger tubes a bit and test after 2-3 h to be safe.
    • Last year starting batches at 12:10, 12:30 pm worked well (w/ lecture going till 1:45 pm), but test cells day before to double-check growth rate.
  2. Put calcium chloride (prep ~8 mL aliquots) on ice.
  3. LB+Amp/Cam plates
  4. LB broth, Amp, Cam
  5. Miniprep solution aliquots
  6. Sequencing primers thawed and diliuted 1:100
  7. Sterile DI water (200μL aliquots)
  8. Thaw NEB buffers and keep on ice, have enzymes at the ready.
  9. For digests: 12 μL parent IPC and 8 μL M124S miniprep for each group.

Day of Lab (R/F):

  • Quiz (prepared by TA).
  • Keep an eye on DE3 densities before and during lab.

Days after Lab (F/Sa, then spring break week):

  • Store plates in fridge, wrapped in parafilm.
  • On M/T, pick two colonies per mutant to grow O/N in liquid culture (Amp+Cam).
  • Also pluck M124S colony per student plate.
  • Prepare DE3/WT-IPC in advance, such that colonies are ready on Mon at the latest.
    • Assuming will need to make 1:20 dilutions next time, need at least 2.4 mL of each
    • Prep 3 (3 mL) O/N tubes to be on safe side

How it went:

  • T/R
    • Sub-cult of O/N to 0.18 OD at 12:15, 12:32. At 1:45+ pm, most at OD 0.8 rather than 0.4-0.6.
    • Day ran quite long for majority of students (5:40 pm), still need to cut some steps for them.
  • W/F
    • Sub-cult of same O/N, after in fridge one night, to 0.18 OD at 12:30 pm. At 1:45+ pm, most on border or a little below 0.4.
    • Day ran slightly long for majority of students (5:15 pm).

Day 5

Materials required:

  1. Put out LB for OD measurements, few mL per group.
  2. Sub-culture each DE3/mutant, 6 mL per tube.
    • Last year, ~1:20 dilution initiated between 10:00 and 10:30 am worked well.
    • This means 4 tubes of mutants per pair.
  3. Also sub-culture enough DE3/wild-type and DE3/M124S for each pair to have one tube (plus make two extra).
  4. Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M.
  5. One-half gel per group, TAE buffer, 1 Kbp and 100 bp ladders available. Put out sample sign.

Day of Lab (T/W):

  • Short quiz.
  • Make sure students turn roller back on!
  • Make sure students measure, then spin down and save at least their -IPTG samples.
  • For recalcitrant +IPTG samples (no colour change), continue induction at RT overnight.

Day after Lab (W/R):

  • Measure the OD (1:10 dilution), then spin down and freeze any +IPTG samples cultured O/N.
    • Post the OD values to the wiki.

How it went:

  • Starting 1:20 sub-culture at 10:30 am (T/R) or even 11:15 am (W/F) gave high log ODs, 1.5 or 1, respectively.
  • More than half the groups had green pellets after 2-2.5 hours of culture.
    • M124S grows more slowly :( Most students had pale yellow pellet, some went with it, some grew O/N. After O/N growth, all pellets were quite green and large.

Day 6

Materials required:

  1. Cell lysis
    • BPER (4 mL aliquots, +40 μL 10% BSA and inhibitors)
    • Lysis enzyme available on ice up front
    • Water (150 μL aliquots)
  2. SDS-PAGE, gel bench
    • Polyacrylamide gels (1 per pair).
    • TGS buffer (1 L per box)
    • Staining boxes, couple of spatulas
    • Coomassie bottle and 50 mL conical tubes for measuring
    • Distilled water in 1 L bottles
  3. SDS-PAGE, ready in hood
    • 2X sample buffer (add 5% β-Me at last minute)
    • Water baths with boiling chips, turn early on in lab
    • Lid locks
    • Waste bottle for stain
  4. Protein purification (see googledoc)
    • Note: prepare solutions ~15% in excess of needed volume
    • Water, Charge Buffer (actually, will probably will buy pre-charged resin this year)
    • Binding Buffer, Wash Buffer, and Elution Buffer w/protease inhibitors
    • Small BSA aliquots ready.
  5. Protein concentration
    • Have 5X Coomassie stain from Bio-Rad, water, tubes ready

Day of Lab:

  • No quiz - a very busy day!
  • Transfer gels to fresh water at end of lab and/or next day.
  • Collect all purified protein samples from students and store at 4 °C.

Day after Lab:

  • Transfer gels to water and take pictures.
  • Put up sign in BPEC reserving Day 7 platereader use.

How it went:

  • This is another long day that may need a bit more black-boxing/efficiency work.

Day 7

Materials required:

  1. Pipetting reservoirs - 2 per group
  2. Calcium solutions - 0.5 mL/soln/group

Day of Lab:

  • Quiz (prepared by TA).
  • Post data to wiki.
  • Instructor teaches the first group of students how to do the multichannel pipetting (avoid bubbles, make sure all tips are actually taking up the same volume, etc.); then TA takes over while instructor is in BPEC for plate reading.

How it went:

  • Protein amounts (fluorescence values) were pretty consistently higher this year than in year's past. In part may be due to using Invitrogen instead of Novagen resin, due to a mix-up; in part may be due to the higher cell ODs for many students. Perhaps in future should aim for high-log rather than mid-log growth, in fact.

Day 8

Materials required:

  1. None: all computer work today.

Day of Lab:

  • Quiz (prepared by TA).

How it went:

  • M124S a much better parallel sample than S101L (run in S09). The change in affinity and cooperativity was dramatic and consistent across the class.
  • The only strange issue is that at very low calcium concentrations the data is noisy rather than simply being a high plateau, or even somewhat consistently starts a little low, then has the high plateau, then proceeds as expected.
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